The use of plasmid DNA as gene transfer vehicle has become widespread in gene therapy. In gene therapy applications, a plasmid carrying a therapeutic gene of interest is introduced into patients; transient expression of the gene in the target cells leads to the desired therapeutic effect.
Recombinant plasmids carrying the therapeutic gene of interest are obtained by cultivation of bacteria. For selecting bacterial transformants and in order to assure maintenance of the plasmids in the bacterial host cell, traditionally, an antibiotic resistance gene is included in the plasmid backbone. Selection for plasmids is achieved by growing the cells in a medium containing the respective antibiotic, in which only plasmid bearing cells are able to grow.
The use of antibiotic resistance genes for selection of plasmids for application in gene therapy is accompanied by severe drawbacks:
Since in gene therapy entire plasmids are being delivered, antibiotic resistance genes are introduced into the treated subject. Although these genes are driven by prokaryotic promoters and are should therefore not be active in mammalian cells and tissues, there is the chance that the delivered genes may be incorporated into the cellular genome and may, if in proximity of a mammalian promoter, become transcribed and expressed.
A second drawback of plasmids that bear antibiotic resistance genes is a potential contamination of the final product with residual antibiotic. In view of possible immune sensitization, this is an issue, especially in the case of beta-lactam antibiotics.
In order to avoid these risks, efforts have been made to ban antibiotic resistance genes from the manufacture of therapeutic plasmids and to develop alternative selection methods.
In an attempt to achieve antibiotic-free selection, plasmids have been used that can compensate a host auxotrophy. However, the main disadvantage of this and all related approaches is that additional genes on the plasmid are required (e.g. Hägg et al., 2004).
Another approach is a concept termed “repressor titration” (Wiliams et al., 1998). According to this concept, a modified E. coli host strain contains the kan gene (kanamycin resistance gene) under the control of the lac operator/promoter. In the absence of an inducer (IPTG or allolactose), the strain cannot grow on kanamycin-containing medium. Transformation with a high copy number plasmid containing the lac operator leads to kan expression by titrating lacI from the operator. Only cells that contain a high plasmid copy number are able to survive after addition of kanamycin. The major drawback of this concept is the fact that, again, the use of antibiotics is indispensable.
It has been an object of the invention to provide a novel system for selection of plasmids that goes without antibiotics.
To solve the problem underlying the invention, the mechanism of replication that is used by plasmids with a ColE1 origin of replication has been exploited. (In the following, plasmids with a ColE1 origin of replication are referred to as “ColE1-type plasmids”.)
A large number of naturally occurring plasmids as well as many of the most commonly used cloning vehicles are ColE1-type plasmids. These plasmids replicate their DNA by using a common mechanism that involves synthesis of two RNA molecules, interaction of these molecules with each other on the one hand and with the template plasmid DNA on the other hand (Helinski, 1996; Kues and Stahl, 1989).
Representatives of ColE1-type plasmids are the naturally occurring ColE1 plasmids pMB1, p15A, pJHCMW1, as well as the commonly used and commercially available cloning vehicles such as pBR322 and related vectors, the pUC plasmids, the pET plasmids and the pBluescript vectors (e.g. Bhagwat and Person, 1981; Balbas et al., 1988; Bolivar, 1979; Vieira and Messing, 1982).
For all these plasmids, ColE1 initiation of replication and regulation of replication have been extensively described (e.g.: Tomizawa, 1981, 1984, 1986, 1989, 1990; Chan et al., 1985; Eguchi et al., 1991a; Cesareni et al., 1991). The ColE1 region contains two promoters for two RNAs that are involved in regulation of replication. Replication from a ColE1-type plasmid starts with the transcription of the preprimer RNA II, 555 bp upstream of the replication origin, by the host's RNA polymerase. During elongation, RNA II folds into specific hairpin structures and, after polymerization of about 550 nucleotides, begins to form a hybrid with the template DNA. Subsequently, the RNA II preprimer is cleaved by RNaseH to form the active primer with a free 3′ OH terminus, which is accessible for DNA polymerase I (Lin-Chao and Cohen, 1991; Merlin and Polisky, 1995).
At the opposite side of the ColE1-type origin strand, RNA I, an antisense RNA of 108 nucleotides, complementary to the 5′ end of RNA II, is transcribed. Transcription of RNA I starts 445 bp upstream from the replication origin, to approximately where the transcription of RNA II starts. RNA I inhibits primer formation and thus replication by binding to the elongating RNA II molecule before the RNA/DNA hybrid is formed.
The interaction of the two RNAs is a stepwise process, in which RNA I and RNA II form several stem loops. They initially interact by base-pairing between their complementary loops to form a so-called “kissing complex”. Subsequently, RNA I hybridizes along RNA II, and a stable duplex is formed. Formation of the kissing complex is crucial for inhibition of replication. As it is the rate limiting step, is has been closely investigated (Gregorian and Crothers, 1995).
Apart from RNA I/RNA II interaction, the rom/rop transcript of ColE1 contributes to plasmid copy number control by increasing the rate of complex formation between RNA II and RNA I. To increase copy number, the gene encoding rom/rop has been deleted on some derivatives of pBR322, for example on pUC19.
The present invention relates, in a first aspect to a non-naturally occurring bacterial cell containing,                i) a DNA sequence encoding a protein, the expression of which is to be regulated, and, operably associated thereto,        ii) a DNA sequence encoding a RNA sequence that mimics a RNA II sequence, or parts thereof, and is complementary to a RNA I sequence that is transcribable from a plasmid with a ColE1 origin of replication.        
In a further aspect, the present invention relates to a host-vector system comprising a plasmid with a ColE1 origin of replication and a bacterial host cell in which said plasmid can be replicated, wherein said host-vector system comprises
a) a plasmid with a ColE1 origin of replication
b) a bacterial host cell in which said plasmid can be replicated, containing,
                i) a DNA sequence encoding a protein, the expression of which is to be regulated, and, operably associated thereto,        ii) a DNA sequence encoding an RNA sequence that mimics an RNA II sequence, or parts thereof, and is complementary to an RNA I sequence that is transcribable from the plasmid a),wherein said RNA sequence defined in ii), in the absence of the plasmid a), allows for expression of said protein andwherein, when said plasmid a) is present inside said host cell b), the RNA I molecule transcribed from the plasmid hybridizes with said RNA sequence defined in ii), whereby expression of said protein is suppressed.        
In a preferred embodiment, the DNA sequence i) is a foreign DNA sequence.
In preferred embodiments, the protein encoded by said foreign DNA i) is toxic or lethal to the host cell.
In a further aspect, the present invention relates to a host-vector system comprising a plasmid with a ColE1 origin of replication and a bacterial host cell in which said plasmid can be replicated, wherein said host-vector system comprises
a) a plasmid with a ColE1 origin of replication,
b) a non-naturally occurring bacterial host cell containing, integrated in its genome,
                i) a foreign DNA sequence encoding a protein that is lethal or toxic to said host cell, and operably associated thereto        ii) a DNA sequence encoding an RNA sequence that mimics an RNA II sequence, or parts thereof, and is complementary to an RNA I sequence transcribable from the plasmid a),wherein said RNA sequence defined in ii), in the absence of the plasmid a), allows for expression of said lethal or toxic protein such that growth of said host cell is completely or partially inhibited andwherein, when said plasmid a) is present inside said host cell, the RNA I molecule transcribed from the plasmid hybridizes with said RNA sequence defined in ii), whereby expression of said lethal or toxic protein is suppressed such that said complete or partial growth inhibition is abrogated in plasmid-bearing cells.        
The invention makes use of the RNA-based copy number control mechanism of ColE1-type plasmids for regulating the expression of one or more genes that are present in the bacterial host cell, preferably inserted in the bacterial genome, and serve as selection markers.
In the following, the DNA sequence of i) (or the RNA transcribed from such DNA, respectively) is referred to as “marker gene” (or “marker RNA”, respectively).
As mentioned above, in an embodiment of the invention, the marker gene encodes a protein that is lethal or toxic per se. In this embodiment, in the meaning of the present invention, the term “marker gene” also encompasses genes the expression of which results in a toxic effect that is not directly due to the expression product, but is based on other mechanisms, e.g. generation of a toxic substance upon expression of the marker gene. For simplicity, in the following, the protein encoded by the marker gene is termed the “marker protein”; in the case that the marker protein is a lethal or toxic protein, it is referred to as “toxic protein”.
In a preferred embodiment, the marker protein is not lethal or toxic per se or due to a toxic effect generated upon its expression, but by repressing the transcription of a gene that is essential for growth of said bacterial cell. Such marker protein, or the DNA encoding it, respectively, is referred to as “repressor” or “repressor gene”, respectively, and the gene that is essential for growth of the bacterial cells is referred to as “essential gene”.
In the following, an RNA sequence that mimics an RNA II sequence, or parts thereof, is referred to as “RNA II-like sequence”.
In the meaning of the present invention “operably associated” means that the DNA sequence i) and the DNA sequence ii) are positioned relative to each other in such a way that expression of the marker protein encoded by said DNA sequence i) is modulated by said RNA sequence ii) (the RNA II-like sequence).
The principle of the invention, i.e. RNA I-mediated marker gene down-regulation or silencing, is shown in FIG. 1:
The RNA II-like sequence is present on the host's transcript in combination with a Shine Dalgarno sequence. The RNA I sequence transcribed from the plasmid functions as an antisense RNA to said RNA II-like sequence and thus inhibits translation of the marker mRNA.
After induction of marker gene expression, in the case that the marker gene encodes a toxic protein, the host can only survive in the presence of the plasmid, because the plasmid provides the RNA I sequence that is complementary to the RNA II-like sequence and therefore hybridizes to the marker gene transcript, thus preventing the translation of the toxic protein. As described, regulation of the system is based on RNA-RNA interaction between the RNA I of the plasmid and, complementary thereto, an RNA II-like sequence of defined length that is positioned upstream or downstream the ribosomal binding site of the marker gene sequence, usually within the host's mRNA.
The length of the RNA II-like sequence and its distance and position relative to the ribosomal binding site and to the start codon of the marker gene must be such that the plasmid-free host is able to translate the mRNA; which means that care must be taken that the RNA II-like sequence does not interfere with ribosomal binding and translation.
Also, the inserted RNA II-like sequence must be designed and positioned such that it guarantees sufficient RNA-RNA interaction of the complementary sequences, so that when the plasmid is present, the RNA I transcribed therefrom binds to the mRNA of the host in an extent sufficient to inhibit translation of the marker gene. Inhibition of the marker gene must be to an extent such that an advantage in growth is provided, as compared to cells where no plasmid, hence no RNA I, is present.
Thus, the bacterial host is engineered such that in the absence of the ColE1-type plasmid the marker mRNA is translated into a marker protein, and in presence of a ColE1-type plasmid, translation of the protein is completely or partially suppressed. In the case that said mRNA encodes a toxic protein that partially or completely inhibits cell growth, hosts that contain the plasmid will survive the toxicity or outgrow plasmid-free hosts.
For the purpose of the present invention, a toxic protein is toxic in the sense that it partially or completely inhibits growth of the cells, at least to an extent to which cells without the marker gene have an advantage with regard to growth rate. If there are two populations of cells, on the one hand a population with the marker gene and, on the other hand, a population without or with an inhibited marker gene, in an equimolar distribution, the cell population without or with an inhibited marker gene will increase to 99% of the population in less than 10 generations.
In an embodiment of the invention, expression of the marker gene is regulated by an additional mechanism, e.g. by induction. Since in the case that the marker gene encodes a toxic protein, the marker gene needs to be turned off during cell propagation, an inducible promoter is advantageously used for transcriptional control, which promotes mRNA transcription only upon addition of an inducer. Examples are the T7 promoter in a T7-polymerase producing host, given that T7-polymerase is under control of the IPTG, or the lactose-inducible Lac-promoter, or an arabinose-inducible promoter.
Alternatively, said marker gene codes for a protein that is not per se toxic, but acts via an indirect mechanism, e.g. an enzyme, which, after addition of a substrate, modifies that substrate to a toxic substance. An example is SacB from Bacillus subtilis. sacB encodes a protein called levan sucrase. This protein turns sucrose into levan, a substance that is toxic to bacteria.
The RNA I sequence of the ColE1-type plasmid represents an essential feature that contributes to the advantages of the system. It provides selection criteria for plasmid-bearing hosts without the use of additional selection markers on the plasmid, e.g. antibiotic resistance genes. Thus, the invention provides an innovative system for antibiotic-free selection of ColE1-type plasmids.
In embodiments of the invention, the following components are useful:
1. Host Cells
Since their replication depends on the host machinery, ColE1-type plasmids are plasmids with a narrow host range. Replication is limited to E. coli and related bacteria such as Salmonella and Klebsiella (Kues and Stahl, 1989). Thus, the only mandatory property of the host is that it has the ability to replicate ColE1 plasmids. Suitable hosts are the widely used Escherichia Coli strains K12 or the B strain or related commercially available strains, e.g. JM108, TG1, DH5alpha, Nova Blue, XL1 Blue, HMS174 or L121 (for review see Casali, 2003).
Preferred genetic features of the host cell are mutations that improve plasmid stability and quality or recovery of intact recombinant protein. Examples of desirable genetic traits are recA (absence of homologous recombination), endA (absence of endonuclease I activity, which improves the quality of plasmid minipreps) or ompT (absence of an outer membrane protease), hsdr (abolished restriction but not methylation of certain sequences), hsdS (abolished restriction and methylation of certain sequences).
In the experiments of the invention, the host strain HMS174(DE3) (Novagen) was used, which contains the DE3 phage with the IPTG inducible T7 polymerase in its genome (Studier and Moffatt, 1986). Another example for a suitable host is HMS174(DE)pLysS, which additionally contains the pACYC184 plasmid (CmR) that carries the gene for the T7-lysozyme to decrease the transcriptional activity of the T7-Promoter in the un-induced state.
Particularly in the case of a lethal marker protein, it is desirable to avoid its expression without induction.
2. Constructs for Engineering the Host Cells
The principle of a construct suitable for engineering the host cells is shown in FIG. 2: All the components—two homologous arms [H], promoter+operator [P+O], RNA I marker sequence (RNA II-like sequence), marker gene [gene] (in the Examples, GFP was used in initial experiments) with a transcriptional terminator and the Kan cassette (kanamycin resistance cassette containing FRT, the +/−FLP recombinase recognition marker sequences; alternatively, other conventional selection markers may be used) are cloned into the multiple cloning site of a suitable vector, e.g. pBluescript KS+. Linear fragments for genomic insertion are cut out with restriction enzymes or amplified by PCR.
The kanamycin resistance cassette can be obtained, by way of example, from the pUC4K vector (Invitrogen). It can be cloned into the fragment at two different sites, namely before or after the marker gene. To avoid unintended premature transcription of the marker gene before it is turned on deliberately, the gene is preferably inserted in the opposite direction of the chromosomal genes.
Preferably, the marker construct is integrated in the bacterial genome. This can be achieved by conventional methods, e.g. by using linear fragments that contain flanking sequences homologous to a neutral site on the chromosome, for example to the attTN7-site (Rogers et al., 1986; Waddel and Craig, 1988; Craig, 1989) or to the recA site. Fragments are transformed into the host, e.g. E. coli strains MG1655 or HMS174 that contain the plasmid pKD46 (Datsenko and Wanner; 2000). This plasmid carries the λ Red function (γ,  exo) that promotes recombination in vivo. Alternatively, DY378 (Yu et al., 2000), an E. coli K12 strain which carries the defective λ prophage, can be used. In case of MG1655 or DY378 the chromosomal locus including the expression fragment can be brought into the HMS174(DE3) genome via transduction by P1 phage. Positive clones are selected for antibiotic resistance, e.g. in the case of using the Kan cassette for kanamycin, or chloramphenicol. The resistance genes can be eliminated afterwards using the FLP recombinase function based on the site-specific recombination system of the yeast 2 micron plasmid, the FLP recombinase and its recombination target sites FRTs (Datsenko and Wanner, 2000).
Alternatively to having the construct integrated in the host's genome, it may be present on a phage or a plasmid that is different from a ColE1-type plasmid and that is compatible with the system of the invention in the sense that it does not influence expression of the marker gene (and the gene of interest). Examples for suitable plasmids or phages are pACYC184 (which is a derivative of miniplasmid p15A; see Chang and Cohen, 1978), R1-miniplasmids (Diaz and Staudenbauer, 1982), F1-based plasmids or filamentous phages (Lin, 1984) or the plasmid pMMB67EH (Fürste et al., 1986) that was used in the experiments of the invention.
More specifically, the elements of suitable constructs can be defined as follows:
2.1. Homologous Arms
It was found in initial experiments of the invention that homologies of 30 bp on either side of the construct are sufficient for recombination by λ Red system (Yu, 2000). However, since better results are obtained with longer homologies, the arms are preferably in the range of 50-400 bp. In the Example homologous arms of 250 and 350 bp are used.
2.2. Promoter
If the foreign marker gene product is per se toxic or lethal to the cell or if it is a repressor, its expression has to be regulated. The promoter region has to contain suitable operator sequences (e.g. the Lac operator) that allow control of gene expression.
According to certain embodiments of the invention, the T7 promoter, the tac or the trc promoter, the lac or the lacUV5 promoter, the pBAD promoter (Guzman et al., 1995), the trp promoter (inhibited by tryptophan), the P1 promoter (with ci repressor) or the gal promoter are used.
When using the lac operator, addition of IPTG (isopropyl thiogalactoside, an artificial inducer of the Lac operon) or lactose are used to activate the marker gene. When an inducible system is used, bacteria are able to survive without induction, but die upon addition of the inducer.
To achieve tight regulation of toxic gene expression, a tightly regulable promoter like the arabinose-inducible PBAD promoter (Guzman et al., 1995) is preferably used, in particular in the case that the marker protein is per se toxic to the cells.
Another way to control expression of the marker gene is by using constitutive promoters in combination with a gene that is non-toxic (e.g. a reporter gene) or only toxic under defined conditions, e.g. the Bacillus subtilis sacB gene. SacB is only toxic to E. coli when sucrose is present.
The promoter is chosen in coordination with the effect of the marker gene product and the required efficiency of down-regulation or silencing effect of RNA I. For example, for a construct containing a non-toxic or less toxic marker gene, a stronger promoter is desirable.
2.3. RNA II-Like Sequence
As RNA I has to act as a partial or complete inhibitor, RNA II-like sequences that are complementary to RNA I (10-555 nt) have to be presented upstream of the marker gene, together with a ribosome binding site (Shine-Dalgarno sequence) that is upstream, downstream or embedded within said RNA II-like sequence. Shine-Dalgarno sequence (SD) refers to a short stretch of nucleotides on a prokaryotic mRNA molecule upstream of the translational start site, that serves to bind to ribosomal RNA and thereby brings the ribosome to the initiation codon on the mRNA. When located upstream of the RNA II-like sequence, the SD sequence, preferably consisting of 7 nucleotides (GAAGGAG) should be located approximately 4 to 15 bp, e.g. 7 bp, upstream of the ATG start codon of the marker gene. In the case that a ribosome binding site is embedded within the RNA I sequence complementary to the marker gene, this sequence should be inserted such that only the stem region is altered, loop structures and preferably the whole secondary structure should stay intact in order to allow antisense RNA interaction with RNA I and formation of a kissing complex.
In an embodiment that provides a start codon in front of the RNAII-like sequence, the construct results in a fusion product comprising the marker sequence and the RNA II-like sequence.
In another embodiment, the RNA II-like sequence is inserted between the ribosomal binding site and the start codon; this approach is limited to the maximal gap possible to allow translation, e.g. 15 to 20 bp. (If the distance between the ribosomal binding site and the start codon increases, translational efficiency decreases.)
Alternatively to directly fusing the RNA II-like sequence and the marker gene, the RNA II-like sequence can be translationally coupled with the marker gene. To achieve this, by way of example, a construct may be used that starts with a start ATG, followed by the RNA II-like sequence, a further ribosome binding site, a sequence which represents an overlap between a stop and a start codon, e.g. TGATG, and the marker sequence. In this case the marker gene is only translated when the RNA II-like sequence has been translated before and separately from the marker gene. The advantage of this set up is that protein fusion to the marker gene is not required. This approach provides the option of separate translation, which may be beneficial for some marker proteins, e.g. in the case of some repressors like the Tet repressor.
Since even single RNA I/RNA II stem loops form kissing complexes (Eguchi 1991b; Gregorian, 1995), it has to be ensured that at least a single loop is formed. In any case, both requirements, i.e. on the one hand translation of the marker mRNA in spite of inserted loop structures and, on the other hand, efficient RNA-RNA antisense reaction between the inserted loop structure of the RNA II-like sequence and the complementary RNA I on the plasmid are fulfilled.
The interaction between RNA I and the marker mRNA that contains the RNA II-like sequence has the purpose to inhibit binding of the ribosome, thereby abolishing translation. Said mRNA is under control of an inducible promoter (e.g. the lac, arabinose or T7 promoter) and after induction (e.g. by IPTG, lactose, arabinose), expression of said marker gene is down-regulated, whenever sufficient RNA I is produced from the plasmid's origin of replication. Preferably, the marker gene encodes a lethal protein or a toxic protein that inhibits cell growth at least to a certain extent (as defined above); in this case, expression results in cell death or decreased cell growth (in plasmid-free cells), whereas down-regulation provides cell-growth (in plasmid-bearing cells).
Alternatively to marker genes that encode lethal or toxic proteins, the marker gene may encode any protein the expression of which is to be regulated during growth of bacterial cells, for whatever purpose. In particular, the marker gene may be a reporter gene, as described below (2.4.).
In the system of the present invention, RNA I, which is normally responsible for down-regulation of plasmid replication, acts as “gene-silencer”, while inhibition of replication is decreased. Thus, the use of the system of the present invention results in an increase of plasmid replication, which is beneficial for survival of the bacterial host cells.
2.4. Marker Gene
RNA I-mediated down-regulation of the marker gene, which is a key feature of the invention, can be applied to any gene the expression of which, for any given purpose, is to be regulated.
According to a first aspect, RNA I-mediated down-regulation is useful for marker genes that are conditionally lethal to the host (e.g. see Davison, 2002, for review).
Examples for marker genes that are toxic per se and suitable in the present invention are genes encoding restriction nucleases (e.g. CviAII, a restriction endonuclease originating from Chlorella virus PBCV-1; Zhang et al., 1992), EcoRI (Torres et al., 2000), genes encoding toxins that interact with proteins, e.g. streptavidin or Stv13 (a truncated, easy soluble streptavidin variant), as described by Szafransky et al., 1997; Kaplan et al., 1999; Sano et al., 1995, which act by deprivation of biotin, an essential protein in cell growth); genes encoding proteins that damage membranes (the E gene protein of ΦX174 (Ronchel et al., 1998; Haidinger et al., 2002), gef(Jensen et al., 1993; Klemm et al., 1995), relF (Knudsen et al., 1995); genes that encode other bacterial toxins, e.g. the ccdb gene (Bernard and Couturier, 1992) that encodes a potent cell killing protein from the F-plasmid trapping the DNA gyrase or sacB from Bacillus Subtilis (Gay et al., 1983); or genes that encode eukaryotic toxins that are toxic to the bacterial host (e.g. FUS; Crozat et al., 1993). When using toxic genes, it is essential that their expression can be modulated by an inducible promoter. This promoter must not be active without inductor, but provide expression upon induction, sufficient to inhibit cell growth.
Further examples of genes toxic in bacteria and useful in the present invention are given by Rawlings, 1999.
In certain embodiments, the marker gene is selected from genes encoding restriction nucleases, streptavidin or genes that have an indirect toxic effect, e.g. SacB, as described above.
In a preferred embodiment, the toxic marker protein is not lethal or toxic per se or due to a toxic effect upon its expression, but a repressor protein which acts by repressing the transcription of a gene that is essential for growth of said bacterial cell.
In this embodiment of the invention, RNA I-mediated down-regulation in the presence of the plasmid affects the repressor. This means that the presence of RNA I and its interaction with the repressor mRNA (the RNA II-like sequence) leads to inhibition of the repressor and thus to activation or up-regulation of an essential gene, with the effect that growth of the cells only occurs in the presence of the replicating plasmid. In this embodiment, the promoter of an essential gene is modified by providing a binding DNA sequence (an “operator”), preferably the natural promoter is replaced by a complete, inducible promoter (containing an operator sequence) in such way that the expressed repressor protein, e.g. the Tet repressor, can bind to that operator, thereby inhibiting transcription and regulating expression of the essential gene, e.g. murA (by expression of the Tet repressor.).
The operator is a DNA sequence to which its specific repressor or enhancer is bound, whereby the transcription of the adjacent gene is regulated, e.g. the lac operator located in the lac promoter with the sequence TGGAATTGTGAGCGGATAACAATT (SEQ ID NO: 53; Gilbert and Maxam, 1973) or derivatives thereof (Bahl et al., 1977). The repressor gene, which should not be present in the wild-type host, is engineered into the genome under the control of an inducible promoter, e.g. the T7, the lac or the tac promoter. Under normal growth conditions, the repressor is not expressed. After induction, by e.g. IPTG, the repressor is expressed, binds to the artificially introduced operator within the promoter region of the essential gene or the artificially inserted promoter and thus inhibits expression of the respective essential gene. Whenever there is replicating ColE1 plasmid present in the host, RNA I is produced which can bind to the repressor mRNA, which had been modified accordingly. By this RNA-RNA interaction, the translation of the repressor is inhibited (analogously to any other toxic marker protein). Consequently, the essential gene product can be produced and the cells maintain viable and grow.
In essence, in this embodiment the bacterial host comprises, besides the RNA II-like sequence, one of its essentials genes (as naturally embedded in the bacterial genome) under the control of an inducible promoter (which has been engineered into the genome to modify or, preferably completely replace the naturally occurring promoter of the essential gene). The promoter region controlling the essential gene also contains a DNA sequence (operator) that is recognized and specifically bound by said repressor protein. The repressor gene, which is engineered into the bacterial chromosome, is also under the control of an inducible promoter that is different from the promoter controlling the essential gene in thus independently inducible.
Essential bacterial genes are known from the literature, e.g. from Gerdes et al., 2002 and 2003, and from the PEC (Profiling the E. coli Chromosome) database (http://www.shigen.nig.ac.jp/ecoli/pec/genes.jsp), e.g. Isoleucyl-tRNA synthetase (ileS), cell division proteins like ftsQ, ftsA, ftsZ, DNA polymerase III alpha subunit (dnaE), murA, map, rps A (30s ribosomal protein S1), rps B (30s ribosomal protein S2), lyt B (global regulator), etc.
A repressor is a protein that binds to an operator located within the promoter of an operon, thereby down-regulation transcription of the gene(s) located within said operon. Examples for repressors suitable in the present invention are the tetracyclin repressor (tet) protein TetR, which regulates transcription of a family of tetracycline resistance determinants in Gram-negative bacteria and binds to tetracyclin (Beck, et al., 1982; Postle et al., 1984), the tryptophan repressor (trp), which binds to the operator of the trp operon, which contains the tryptophan biosynthesis gene (Yanofski et al., 1987).
Examples for inducible promoters are promoters, where transcription starts upon addition of a substance, thus being regulable by the environment, e.g. the lac promoter, which is inducible by IPTG (Jacob and Monod, 1961), the arabinose-promoter (pBAD), inducible by arabinose (Guzman et al., 1995), and copper-inducible promoters (Rouch and Brown, 1997).
In the experiments of the invention, the tet-repressor (tetR) was chosen to be the repressor gene, which served as “toxic” marker gene by turning off an essential bacterial gene upon addition of the inducer IPTG.
For implementation of the repressor gene approach, two types of cassettes are designed and inserted in the bacterial chromosome in the experiments of the invention (Example 4). The first set of constructs comprises cassettes that serve to replace (or modify) promoters of specific essential genes on the genome. The second type of cassettes serve as test constructs employing GFP as a surrogate for an essential gene to provide proof of concept. The aim of the experiments using the GFP test constructs is to evaluate regulatory cascades, promoter strengths and thus adjustment of all interacting components of the system.
Thus, in another embodiment, the marker gene is a reporter gene, e.g. encoding GFP (Green Fluorescent Protein), hSOD (human superoxide dismutase), CAT (chloramphenicol acetyltransferase) or luciferase.
A reporter gene is useful in cultivation processes whenever information on the presence or absence of a ColE1-type plasmid in a host cell or on plasmid copy number is needed. Such information is particularly useful when fermentation processes are to be optimized with regard to control of plasmid copy number.
A reporter gene may also serve as a surrogate of a toxic marker gene, and may thus be used in experimental settings that aim at proving the functionality of constructs to be employed for the gene-regulating or silencing and to determine their effect on a toxic marker gene.
In order to evaluate the functionality of constructs designed for engineering a bacterial host such that expression of a toxic marker gene can by regulated by a ColE1-type plasmid, the reporter gene “green fluorescent protein” (GFP) served as a model in the initial experiments of the invention. Due to its auto-fluorescence (Tsien, 1998) GFP was considered suitable to substitute the marker gene, or the essential gene, respectively, in the initial experiments.
In certain embodiments of the invention, the marker gene may be an endogenous host gene, which may be any gene of interest that is intended to be regulated. In this case, the host cell is engineered such that the sequence encoding the RNA II-like sequence is operably associated with the relevant host gene, as described in 2.3.
3. ColE1-Type Plasmid
In the present invention, all ColE1-type plasmids with their natural RNA I/RNA II pairs, as well as with modified RNA I and/or RNA II sequences, e.g. as described in WO 02/29067, may be used.
As mentioned above, representatives of useful ColE1-type plasmids are the naturally occurring ColE1 plasmids pMB1, p15A, pJHCMW1, as well as the commonly used and commercially available cloning vehicles such as pBR322 and related vectors, the pUC plasmids, the pET plasmids and the pBluescript vectors.
No antibiotic resistance genes need to be included in the plasmid sequence. As essential elements, the plasmid basically only contains the ColE1 origin of replication and the gene expression cassette carrying the gene of interest.
The gene of interest on the plasmid and its promoter depend on the type of application; the invention is not limited in any way with respect to the gene of interest, e.g. a therapeutic gene. For gene therapy applications, the gene may be operably associated to an eukaryotic promoter, e.g. the CMV promoter.